EMDB/PDB/SASBDBから引用されている文献のデータベース

キーワード
構造解析手法	All X線回折 NMR 電子顕微鏡小角散乱中性子線回折線維回折粉末回折赤外分光 EPR FRET 理論モデルハイブリッド
著者・登録者
ジャーナル
IF	>30 >20 >10 >5 all

タイトル	Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins.
ジャーナル・号・ページ	Nat Protoc, Year 2024
掲載日	2024年4月4日
著者	Rebeccah A Warmack / Belinda B Wenke / Thomas Spatzal / Douglas C Rees /
PubMed 要旨	Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast ...Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.
リンク	Nat Protoc / PubMed:38575747
手法	EM (単粒子)
解像度	2.12 - 2.57 Å
構造データ	27317 8dby EMDB-27317, PDB-8dby: CryoEM structure of anaerobically prepared nitrogenase MoFe-protein on ultrathin carbon 手法: EM (単粒子) / 解像度: 2.26 Å 27404 8dfc EMDB-27404, PDB-8dfc: CryoEM structure of the 1:1 ADP-tetrafluoroaluminate stabilized nitrogenase complex from Azotobacter vinelandii 手法: EM (単粒子) / 解像度: 2.48 Å 27405 8dfd EMDB-27405, PDB-8dfd: CryoEM structure of the 2:1 ADP-tetrafluoroaluminate stabilized nitrogenase complex from Azotobacter vinelandii 手法: EM (単粒子) / 解像度: 2.12 Å 41151 8tc3 EMDB-41151, PDB-8tc3: CryoEM structure of nucleotide-free form of the nitrogenase iron protein from A. vinelandii 手法: EM (単粒子) / 解像度: 2.57 Å
化合物	ChemComp-ICS: iron-sulfur-molybdenum cluster with interstitial carbon ChemComp-HCA: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / (2R)-2-ヒドロキシ-1,2,4-ブタントリカルボン酸 ChemComp-FE: Unknown entry / 鉄 ChemComp-CLF: FE(8)-S(7) CLUSTER ChemComp-HOH: WATER / 水 ChemComp-SF4: IRON/SULFUR CLUSTER / 鉄・硫黄クラスター ChemComp-ADP: ADENOSINE-5'-DIPHOSPHATE / ADP / ADP, エネルギー貯蔵分子YM / アデノシン二リン酸 ChemComp-ALF: TETRAFLUOROALUMINATE ION / テトラフルオロアルミナ-ト ChemComp-MG*: Unknown entry / マグネシウムジカチオン
由来	azotobacter vinelandii (窒素固定) azotobacter vinelandii dj (窒素固定) discosoma sp. lw-2004 (イソギンチャク)
キーワード	METAL BINDING PROTEIN / OXIDOREDUCTASE (酸化還元酵素) / Nitrogenase (ニトロゲナーゼ) / metalloenzyme (金属タンパク質) / ATPase (ATPアーゼ) / metalloprotein (金属タンパク質) / iron-sulfur (鉄硫黄タンパク質)